PROJECT SUMMARY Ductal Carcinoma in situ (DCIS) and Atypical Ductal Hyperplasia (ADH) are the most common forms of early- stage breast cancer and are non-obligatory precursors to invasive ductal carcinoma (IDC). Genomic studies of tumor initiation and invasion in premalignant breast cancer represents a major gap in knowledge and have been challenging due to several technical barriers, including the limited number of tumor cells in the ducts, the logistical challenge of obtaining fresh or frozen tissues, and the lack of spatial resolution in standard genomic methods. To overcome these challenges, we have developed cutting-edge single cell sequencing methods to profile DNA, RNA and Epigenomics with spatial-resolution in human tissue sections. We have also established a unique fresh DCIS tissue collection program at MD Anderson to procure viable cells from DCIS patients directly after surgery for single cell sequencing to overcome logistical challenges in sample sources. In our previous work, we applied these methods to study copy number evolution during invasion in synchronous DCIS-IDC patients, which identified punctuated copy number evolution (PCNE) in the ducts and revealed a multi-clonal model of invasion (Casasent et al. 2018, Cell). This proposal will greatly extend these initial studies to study mutational, epigenomic and transcriptional reprogramming in the tumor cells and the microenvironment during premalignant breast cancer progression. Our central hypothesis is that the evolution of premalignant lesions requires initiating mutations and punctuated copy number evolution (PCNE), which is followed by transcriptional and epigenomic reprogramming in tumor cells and the microenvironment that leads to invasion. AIM1 will investigate breast tumor initiation in ADH and DCIS, while AIM2 will focus on tumor invasion in DCIS and IDC, and AIM3 will study role of the tumor microenvironment in DCIS progression. Candidate mutations identified in these aims will be functionally validated in vitro. Completion of these aims will greatly improve our fundamental knowledge of tumor initiation and invasion in premalignant breast cancer progression, which may lead to new biomarkers and diagnostic modalities for identifying ADH and DCIS patients that will progress to invasive disease and is critical unmet clinical need. Our long-term goal is to translate single cell sequencing technologies into the clinic, where they are poised to make a major impact on the diagnosis and treatment of premalignant breast cancer patients. The proposed aims are directly aligned with the mission of NIH to reduce morbidity and improve the quality of life for breast cancer patients through early detection.